The goal of this project is to elucidate structure-function relationships in macromolecular assemblies. During FY17, our studies focused on retinoschisin (RS1), a junctional protein in the human retina; encapsulin, a bacterial nanocompartment that sequesters iron; and computational tools used in image analysis. 1) RS1 is a protein required to maintain the structural and functional integrity of the retina. Mutations in RS1 lead to early vision impairment in young males, a condition termed X-linked retinoschisis (XLRS). From earlier work, RS1 was thought to form an octamer, with each subunit comprising a discoidin domain (DS) and a small N-terminal domain (RS1 domain). We used cryo-EM to determine the structure of RS1 at 0.4 nm resolution, finding that the complex consists, in fact, of two apposed octameric rings. The RS1 domains occupy the centers of the rings, but are less clearly defined, suggesting mobility. We combined the cryo-EM density map with crystal structures of other discoidin domains to create a high-resolution model of the double octamer. This model is consistent with intramolecular and intermolecular disulfides previously reported. The interfaces internal to and between rings accommodate residues implicated in XLRS, indicating the importance of correct assembly of the 16-meric complex to obtain a correctly constituted junction. We published a paper reporting this analysis in May 2016 (G. Tolun et al., Proc Natl Acad Sci USA 113:5287-92). Since then, the project has been extended as follows. From the known sites of disease-causing RS1 mutants, adhesive functionality appears to be associated with the spikes located at the periphery of the rings. RS1 is thought to interact with cell membranes by binding to lipid head-groups, via membrane proteins and/or carbohydrate moieties on glycolipids or glycoproteins. In particular, RS1 has been shown to bind to galactose. Accordingly, we performed cryoEM on RS1 with galactose bound. To our surprise, we found that the double rings form long branched chains, constituting a 2D network. In these chains, the molecules mostly present side-views, suggesting that they may interact with the air-water interface through their spikes. The interface is thought to be a mimic for lipid bilayers. Within chains there are spike-spike interactions between neighboring molecules. The spikes are also involved in branching interactions, where they mostly interact with the tops of the rings on another molecule. The ability of RS1 to form such a network suggests that in situ it may be forming a 3D scaffold between photoreceptors to glue them together. A paper reporting this phase of the project is in preparation. 2) Encapsulin is a virus capsid-like nanocompartment that sequesters iron, thereby protecting bacteria from oxidative stress. In earlier work reported in FY15, we characterized the structure of encapsulin isolated from the Gram-negative bacterium Myxococcus xanthus. This particle has a protein shell assembled from 180 copies of EncA protein, and smaller amounts of three internal proteins (EncB; EncC; EncD). Using cryo-EM, we showed that EncA assembles into an icosahedral capsid 32 nm in diameter with a triangulation number of T=3. Our analysis showed that EncA has the fold first observed in bacteriophage HK97 capsid. Native nanocompartments have dense iron-rich cores. Functionally, they resemble ferritins, but with a massively greater capacity (30,000 Fe atoms vs. 3,000 in ferritin). In FY17, our main thrust has been an attempt to seek high resolution cryo-EM data on particles of the purified internal protein ClpB. The data obtained to date are encouraging in the sense that EncB makes quite large particles, enhancing the feasibility of this approach, but progress has been hampered by overly crowded grids and eventual heterogeneity of the particles. 3) Development of image processing software for three-dimensional electron microscopy. Bsoft is a comprehensive suite of computer programs for image processing of cryo-EM images and cryo-ET data that is maintained, disseminated, and further developed in the LSBR by B. Heymann. In FY17, an updated and upgraded version of Bsoft (Bsoft 2.0.0) was released. In it, the code structure has been modified to eliminate legacy libraries and to introduce a more general compilation scheme. The intention is to develop along more modern standards with coding in C++ to ensure better stability and longevity. The single particle analysis (SPA) capabilities have been expanded to allow better 2D analysis and classification. Specifically, the handling of dose-fractionated movies (motion correction) has been improved; the processing of tomographic tilt series can now be done through the Bshow interface without the need to write command lines. This includes estimation of the contrast transfer function parameters and correcting for it during reconstruction. In the last few years, advances in cryo-EM and image processing have made possible density maps at resolutions comparable to those achieved by X-ray crystallography. This development has raised the question of whether any particular work-flows or processing strategies achieve the best results and/or whether artifacts may be introduced in some circumstances. To this end, B. Heymann is participating in the Map Challenge project intended to assess processing and map validation (http://challenges.emdatabank.org/?q=2015_map_challenge). A meeting to discuss the results and conclusions is scheduled at Stanford University, Oct 5-8, 2017. The conclusions will be published.